Lysosome-targeted light-controlled fluorescent molecular switch and its synthesis and application

Abstract

The present invention relates to the field of lysosome imaging probes and optical super-resolution imaging. A light-controlled fluorescent molecular switch targeting lysosomes has a structural formula as shown in Formula I: wherein R is any one of NMe2 and NEt2.

Description

Technical Field

[0001] The present invention relates to the fields of lysosome imaging probes and optical super-resolution imaging, and in particular to lysosome super-resolution imaging. Background Art

[0002] Lysosomes are important acidic organelles within cells that contain a variety of hydrolases and various organic substances. Lysosomes play a vital role in digesting, breaking down, and recycling intracellular metabolites and are involved in a variety of life processes. When lysosomes become dysfunctional, they can lead to a variety of diseases, including a range of lysosomal storage diseases, neurodegenerative diseases, cardiovascular diseases, as well as inflammation and cancer. Lysosomes move at high speeds within cells to meet the cell's ever-changing needs. When cells are in a state of nutrient deficiency, they can move rapidly and close to the nucleus. Monitoring the dynamic movement of lysosomes has profound implications for understanding intracellular metabolism, microenvironmental changes, and the workings of lysosomes.

[0003] Optical imaging technology is an important tool for studying various life activities. However, traditional fluorescence imaging technology cannot meet higher imaging requirements due to the existence of optical diffraction limits. Super-resolution microscopy technology developed in recent years has broken through the diffraction limit in optical imaging and increased the resolution of traditional imaging by 10 to 20 times. For example, the spatial resolution of stochastic optical reconstruction microscopy (STORM) based on single-molecule localization can reach 20 nanometers. Super-resolution microscopy technology based on single-molecule localization requires dyes with fluorescence "on-off" functions to achieve single-molecule detection and localization. However, the STORM imaging probes currently used in living cells are severely limited by photobleaching and phototoxicity, making it challenging to achieve long-term super-resolution imaging of living cells. Therefore, the development of probes that use low-power visible light for long-term STORM imaging is an urgent need in the field of super-resolution imaging. Summary of the Invention

[0004] The purpose of the present invention is to provide a light-controlled fluorescent molecular switch targeting lysosomes to solve the above technical problems.

[0005] Another object of the present invention is to provide a method for synthesizing a lysosome-targeted light-controlled fluorescent molecular switch to prepare the lysosome-targeted light-controlled fluorescent molecular switch.

[0006] Another object of the present invention is to provide an application of a lysosome-targeted light-controlled fluorescent molecular switch.

[0007] The technical problem solved by the present invention can be achieved by adopting the following technical solutions:

[0008] A light-controlled fluorescent molecular switch targeting lysosomes, with the structural formula shown in Ⅰ.

[0009]

[0010] In formula I, R is independently selected from NMe2, NEt2, Any one of .

[0011] The ethene bridge described in structural formula I represents an ethylene bridge, which is any group in formula II.

[0012]

[0013] A method for synthesizing a lysosome-targeted light-controlled fluorescent molecular switch, the synthesis route is as follows:

[0014]

[0015] The specific steps are:

[0016] Step 1: dissolve 1,2-bis(2-ethyl-1-benzothiophen-3-yl)perfluorocyclopentene and N-bromosuccinimide in dichloromethane at a molar ratio of 1:2.1, stir at room temperature in the dark for 20 hours, and then spin-dry the solvent. The mixture is further separated by column chromatography and purified to obtain a brominated product.

[0017] Step 2: Dissolve the product in step 1 and m-chloroperbenzoic acid in dry dichloromethane at a molar ratio of 1:12, stir at room temperature for 12 hours under a nitrogen atmosphere, add water to quench the reaction, extract with dichloromethane, evaporate the solvent under reduced pressure, and separate and purify by column chromatography to obtain the oxidation product.

[0018] Step 3: The product 4-(4-pyridyl)phenylboronic acid pinacol ester in step 2 was dissolved in tetrahydrofuran in a molar ratio of (1:2.5), and an aqueous solution of potassium carbonate was added. Tetrakistriphenylphosphine palladium (5% wt) was added under a nitrogen atmosphere and stirred for 5 hours. The tetrahydrofuran solvent was dried and extracted with dichloromethane. The solvent was evaporated under reduced pressure and the final product was separated and purified by column chromatography.

[0019] Lysosome-targeted light-controlled fluorescent molecular switch can be used as a STORM imaging probe for lysosome targeting. Experimental results show that the absorption spectrum of the lysosome probe of the present invention extends to the visible light region, and the fluorescence is near-infrared emission. The full visible light can be used for light switching reaction, avoiding the damage of ultraviolet light to living cells. In addition, this probe is insensitive to acidic environments, which is conducive to long-term imaging inside acidic lysosomes. The above-mentioned probe has an excellent lysosome targeting effect and has good anti-photobleaching properties compared with the commercial dye lysosome green. The super-resolution STORM imaging results in living cells show that long-term monitoring of the dynamic movement of lysosomes can be achieved using a low-power (0.75mW) 546nm laser. The lysosome-targeted STORM imaging probe of the present invention can be applied to dynamic super-resolution imaging of lysosomes in living cells.

[0020] In addition, lysosome-targeted light-controlled fluorescent molecular switches can also be used in fields such as sensing and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings in the specification, which constitute a part of this application, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 , is the H NMR spectrum of the fluorescent probe PM.

[0023] Figure 2 , is the NMR carbon spectrum of the fluorescent probe PM.

[0024] Figure 3 , is the high-resolution mass spectrometry of the fluorescent probe PM.

[0025] Figure 4 , is a fluorescent probe PM in a tetradioxane solvent (concentration 10 -5 UV-visible absorption spectrum of M).

[0026] Figure 5 , is a fluorescent probe PM in a tetradioxane solvent (concentration 10 -5 M) fluorescence spectrum.

[0027] Figure 6 , are the fluorescence spectra of the fluorescent probe PM in PBS buffer solutions of different pH values.

[0028] Figure 7 , Confocal images of cultured HeLa cells co-stained with the fluorescent probe PM (5 μM) and a commercial lysosomal marker dye (LTG, 0.1 μM).

[0029] Figure 8 , confocal images of cultured HeLa cells stained with fluorescent probe PM (5 μM) and commercial lysosome marker dye (LTR, 0.1 μM) under 540 nm illumination over time.

[0030] Figure 9 , are super-resolution STORM images of HeLa cells after fixation and cultured and stained with the fluorescent probe PM (5μM).

[0031] Figure 10 , dynamic super-resolution STORM images of cultured HeLa cells stained with the fluorescent probe PM (5μM). DETAILED DESCRIPTION

[0032] The present invention will be further described below by way of examples, the purpose of which is merely to provide a better understanding of the present invention. Therefore, the examples given do not limit the scope of protection of the present invention.

[0033] Example 1

[0034] The synthetic route and product structure of the fluorescent probe PM are as follows:

[0035]

[0036] The synthesis steps are as follows:

[0037] Step 1. P-1 (500.0 mg, 1.1 mmol), N-bromosuccinimide (411.2 mg, 2.3 mmol) and dichloromethane (50.0 mL) were added to a 150 mL single-necked flask. The mixture was stirred at room temperature in the dark for 20 hours, and then the solvent was removed by distillation under reduced pressure. The white intermediate P-1 (550.5 mg) was separated by column chromatography, with a yield of 83.7%.

[0038] Step 2: P-2 (500.0 mg, 0.8 mmol), m-chloroperbenzoic acid (1.6 g, 9.6 mmol), and dichloromethane (50.0 mL) were added to a 150 mL single-necked flask and reacted at room temperature for 10 h under nitrogen. The reaction was quenched by adding water. After extraction with dichloromethane and removal of the solvent under reduced pressure, the product was purified by column chromatography to obtain 410.2 mg of P-3 as a pale yellow solid in a 74.6% yield.

[0039] Step 3. P-3 (300.0 mg, 0.4 mmol), 4-(4-pyridyl)phenylboronic acid pinacol ester (280.3 mg, 1.0 mmol), potassium carbonate (138.2 mg, 1.0 mmol), tetrahydrofuran (20.0 mL) and water (4.0 mL) were added to a 150 mL single-necked flask, and tetrakistriphenylphosphine palladium (5% wt) was added under a nitrogen atmosphere and stirred for 5 hours. The tetrahydrofuran solvent was dried and extracted with dichloromethane. The solvent was evaporated under reduced pressure and purified by column chromatography to obtain 200.6 mg of a dark green solid PM with a yield of 54.1%.

[0040] The powder products were characterized by H NMR, C NMR and high resolution mass spectrometry. Figure 1 、 Figure 2 、 Figure 3shown. ap:p=61:39.1H NMR(400MHz, CDCl3), δ7.93(s,1.0H,ap),7.85(s,0.6H,ap),7.76(d,J=8.0Hz,1H,ap),7.58(d,J=8.0Hz,0.6 H,p),7.53(d,J=8.4Hz,2H,ap),7.44(d,J=8.4Hz,1.3H,ap),7.22(d,J=8.4Hz,1H,ap),7.17(d,J=8.4Hz,0.6H ,ap),6.99(d,J=8.4Hz,2H,ap),6.94(d,J=8.4Hz,1.3H,ap),3.88(m,6.6H,ap / p),3.28-3.19(m,6.5H,ap / p) ,2.70-2.50(m,2.5H,ap),2.46-2.35(m,1.2H,p),1.42(t,J=7.6Hz,2.1H,p),1.07(t,J=7.7Hz,3.3H,ap).13C NMR (151MHz, CDCl3) δ147.80,147.42,143.99,143.86,136.49,136.45,131.02,130.69,127.92,127.90,127.0 6,123.53,123.10,122.88,120.13,115.68,66.70,66.66,48.65,19.23,19.09,11.98,11.71.High-resolution mass spectrometry(ESIpositive ion mode for[M+Na] + ):Calcd.for C 45 H 40 F6N2O6S2:905.2124; Found:905.2098.

[0041] Example 2

[0042] The probe PM was dissolved in 1,4-dioxane solvent (concentration of 10 -5 M), and the UV-visible absorption spectrum under 365 nm UV light ( Figure 4 ) and fluorescence spectra ( Figure 5 The results showed that the absorption peak of the open-ring PM extends to 500 nm, with a distinct absorption band in the visible light region. The maximum absorption peak of the closed-ring PM is located at 550 nm. The open-ring state does not fluoresce, while the closed-ring state emits near-infrared fluorescence.

[0043] Example 3

[0044] The probe PM was dissolved in PBS buffer solutions with different pH values ​​(concentration of 10 -5 M), and measured the fluorescence spectra of PM in different pH solutions ( Figure 6 The results showed that the fluorescence intensity of PM was not affected by the pH of the solution, indicating that the PM probe has acid-resistant properties.

[0045] Example 4

[0046] HeLa cells were incubated with PM (5 μM) and a commercial lysosomal marker (LTG, 0.1 μM) for 20 minutes. Fluorescence staining was observed in two channels using confocal fluorescence microscopy. The green channel, derived from the commercial lysosomal marker, was excited at 546 nm and captured fluorescence signals in the 600-700 nm range. The red channel, derived from the PM dye, was excited at 546 nm and captured fluorescence signals in the 650-750 nm range. Intensity distribution across the entire cell's linear region of interest (ROI) showed good overlap between PM and the commercial lysosomal marker, demonstrating excellent colocalization. These results demonstrate that the PM probe exhibits excellent lysosomal localization.

[0047] Example 5

[0048] Hela cells were incubated with PM (5 μM) and commercial lysosomal marker dye (LTR, 0.1 μM) for 20 min, and the fluorescence signals in the two channels were observed under 546 nm continuous light by fluorescence confocal microscopy ( Figure 7 a) Set the same excitation wavelength and fluorescence signal collection band for the two dyes (excitation wavelength is 546nm, and fluorescence signal is collected in the 600-700nm band). Comparative observation shows that after 30 minutes of 546nm light exposure, the fluorescence signal intensity in the lysosomes stained by PM is 80% of the initial value, while the fluorescence signal intensity in the lysosomes stained by commercial lysosomal probe is 15% of the initial value ( Figure 7 b), The results indicate that PM dyes have good resistance to photobleaching and are suitable for long-term lysosomal imaging.

[0049] Example 6

[0050] Hela cells were incubated with PM (5 μM) for 20 min and fixed with 4% paraformaldehyde. Super-resolution images of lysosomes were captured using a Nikon STORM microscope. Only a 546 nm laser (power 0.75 mW) was used during the experiment, and 10,000 frames were captured. The experimental results showed that compared with traditional fluorescence images, super-resolution STORM images had a significant improvement in resolution ( Figure 8 ), the PM probe can be used as an excellent super-resolution imaging reagent for lysosomes.

[0051] Example 7

[0052] Hela cells were incubated with PM (5 μM) for 20 minutes, and dynamic super-resolution images of lysosomes in living cells were captured using a Nikon STORM microscope. The experimental results showed that the movement of lysosomes in cells could be clearly observed through super-resolution imaging, and the probe PM has the potential to be used as a super-resolution dynamic imaging agent for lysosomes in living cells.

Claims

1. A light-controlled fluorescent molecular switch targeting lysosomes, characterized by: Its structural formula is shown in Formula I: In formula I, R is , Any one of; the Ethene bridge represents an ethylene bridge, which is the following group: .

2. A method for synthesizing a light-controlled fluorescent molecular switch targeting lysosomes, characterized in that: 。 3. The method for synthesizing a lysosomal-targeted light-controlled fluorescent molecular switch according to claim 2, wherein: 。 4. A method for synthesizing a light-controlled fluorescent molecular switch targeting lysosomes, characterized by: The steps include: Step 1, dissolving 1,2-bis(2-ethyl-1-benzothiophen-3-yl)perfluorocyclopentene and N-bromosuccinimide in dichloromethane at a molar ratio of 1:2.1, stirring at room temperature in the dark for 20 hours, and then drying the solvent by spin drying. The product is further separated by column chromatography and purified to obtain a brominated product; Step 2: dissolving the product of step 1 and m-chloroperbenzoic acid in dry dichloromethane at a molar ratio of 1:12, stirring at room temperature for 12 hours under a nitrogen atmosphere, adding water to quench the reaction, extracting with dichloromethane, and removing the solvent under reduced pressure, followed by separation and purification by column chromatography to obtain an oxidation product; Step 3, the product of step 2 above, 4-(4-pyridyl)phenylboronic acid pinacol ester, was dissolved in tetrahydrofuran at a molar ratio of 1:2.5, an aqueous solution of potassium carbonate was added, and 5% wt of tetrakistriphenylphosphine palladium was added under nitrogen atmosphere and stirred for 5 hours. The tetrahydrofuran solvent was dried and extracted with dichloromethane. The solvent was evaporated under reduced pressure and the final product was separated and purified by column chromatography.

5. The use of the light-controlled fluorescent molecular switch targeting lysosomes as claimed in claim 1, characterized in that: Preparation of STORM imaging probes targeting lysosomes.

6. The use of the light-controlled fluorescent molecular switch targeting lysosomes as claimed in claim 5, characterized in that: Achieve dynamic super-resolution imaging of lysosomes in living cells.

7. The use of the light-controlled fluorescent molecular switch targeting lysosomes as claimed in claim 5, characterized in that: Used to monitor the rapid movement of lysosomes in living cells.

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